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Cholangiocarcinoma (CCA) is a rare malignancy that develops at any point along the biliary tree. CCA has a poor prognosis, its clinical management remains challenging, and effective treatments are lacking. Therefore, preclinical research is of pivotal importance and necessary to acquire a deeper understanding of CCA and improve therapeutic outcomes. Preclinical research involves developing and managing complementary experimental models, from in vitro assays using primary cells or cell lines cultured in 2D or 3D to in vivo models with engrafted material, chemically induced CCA or genetically engineered models. All are valuable tools with well-defined advantages and limitations. The choice of a preclinical model is guided by the question(s) to be addressed; ideally, results should be recapitulated in independent approaches. In this Consensus Statement, a task force of 45 experts in CCA molecular and cellular biology and clinicians, including pathologists, from ten countries provides recommendations on the minimal criteria for preclinical models to provide a uniform approach. These recommendations are based on two rounds of questionnaires completed by 35 (first round) and 45 (second round) experts to reach a consensus with 13 statements. An agreement was defined when at least 90% of the participants voting anonymously agreed with a statement. The ultimate goal was to transfer basic laboratory research to the clinics through increased disease understanding and to develop clinical biomarkers and innovative therapies for patients with CCA.

Organoids are simple tissue-engineered cell-based in vitro models that recapitulate many aspects of the complex structure and function of the corresponding in vivo tissue. They can be dissected and interrogated for fundamental mechanistic studies on development, regeneration and repair in human tissues, and can also be used in diagnostics, disease modelling, drug discovery and personalized medicine. Organoids are derived from either pluripotent or tissue-resident stem (embryonic or adult) or progenitor or differentiated cells from healthy or diseased tissues, such as tumours. To date, numerous organoid engineering strategies that support organoid culture and growth, proliferation, differentiation and maturation have been reported. This Primer highlights the rationale underlying the selection and development of these materials and methods to control the cellular/tissue niche; and therefore, the structure and function of the engineered organoid. We also discuss key considerations for generating robust organoids, such as those related to cell isolation and seeding, matrix and soluble factor selection, physical cues and integration. The general standards for data quality, reproducibility and deposition within the organoid community are also outlined. Lastly, we conclude by elaborating on the limitations of organoids in different applications, and the key priorities in organoid engineering for the coming years.

This paper encompasses three contributions by industry professionals and university researchers. The contributions describe different trends in automotive products, including both manufacturing test and run-time reliability strategies. The subjects considered in this session deal with critical factors, from optimizing the final test before shipment to market to in-field reliability during operative life.

What will be the most important areas of research in biotech over the coming years? Which technologies will be most important to advance knowledge and applications in these areas? Nature Biotechnology reached out to a set of faculty doing outstanding work in research areas representative of the journal’s remit and asked them to contribute their vision of where their fields are going.

What will be the most important areas of research in biotech over the coming years? Which technologies will be most important to advance knowledge and applications in these areas? Nature Biotechnology reached out to a set of faculty doing outstanding work in research areas representative of the journal’s remit and asked them to contribute their vision of where their fields are going.

Background: Organoids are morphologically heterogeneous three-dimensional cell culture systems and serve as an ideal model for understanding the principles of collective cell behaviour in mammalian organs during development, homeostasis, regeneration, and pathogenesis. To investigate the underlying cell organisation principles of organoids, we imaged hundreds of pancreas and cholangiocarcinoma organoids in parallel using light sheet and bright-field microscopy for up to 7 days. Results: We quantified organoid behaviour at single-cell (microscale), individual-organoid (mesoscale), and entire-culture (macroscale) levels. At single-cell resolution, we monitored formation, monolayer polarisation, and degeneration and identified diverse behaviours, including lumen expansion and decline (size oscillation), migration, rotation, and multi-organoid fusion. Detailed individual organoid quantifications lead to a mechanical 3D agent-based model. A derived scaling law and simulations support the hypotheses that size oscillations depend on organoid properties and cell division dynamics, which is confirmed by bright-field microscopy analysis of entire cultures. Conclusion: Our multiscale analysis provides a systematic picture of the diversity of cell organisation in organoids by identifying and quantifying the core regulatory principles of organoid morphogenesis.

LifeTime aims to track, understand and target human cells during the onset and progression of complex diseases and their response to therapy at single-cell resolution. This mission will be implemented through the development and integration of single-cell multi-omics and imaging, artificial intelligence and patient-derived experimental disease models during progression from health to disease. Analysis of such large molecular and clinical datasets will discover molecular mechanisms, create predictive computational models of disease progression, and reveal new drug targets and therapies. Timely detection and interception of disease embedded in an ethical and patient-centered vision will be achieved through interactions across academia, hospitals, patient-associations, health data management systems and industry. Applying this strategy to key medical challenges in cancer, neurological, infectious, chronic inflammatory and cardiovascular diseases at the single-cell level will usher in cell-based interceptive medicine in Europe over the next decade., QC 20201117Correction in: Nature, Volume 587, Issue 7834, Pages 377 - 386, 19 November 2020. DOI: 10.1038/s41586-021-03287-8, Scopus ID: 2-s2.0-85102698588

LifeTime aims to track, understand and target human cells during the onset and progression of complex diseases and their response to therapy at single-cell resolution. This mission will be implemented through the development and integration of single-cell multi-omics and imaging, artificial intelligence and patient-derived experimental disease models during progression from health to disease. Analysis of such large molecular and clinical datasets will discover molecular mechanisms, create predictive computational models of disease progression, and reveal new drug targets and therapies. Timely detection and interception of disease embedded in an ethical and patient-centered vision will be achieved through interactions across academia, hospitals, patient-associations, health data management systems and industry. Applying this strategy to key medical challenges in cancer, neurological, infectious, chronic inflammatory and cardiovascular diseases at the single-cell level will usher in cell-based interceptive medicine in Europe over the next decade.

BACKGROUND & AIMS: Alagille syndrome is a genetic disorder characterized by cholestasis, ocular abnormalities, characteristic facial features, heart defects, and vertebral malformations. Most cases are associated with mutations in JAGGED1 (JAG1), which encodes a Notch ligand, although it is not clear how these contribute to disease development. We aimed to develop a mouse model of Alagille syndrome to elucidate these mechanisms. METHODS: Mice with a missense mutation (H268Q) in Jag1 (Jag1+/Ndr mice) were outbred to a C3H/C57bl6 background to generate a mouse model for Alagille syndrome (Jag1Ndr/Ndr mice). Liver tissues were collected at different timepoints during development, analyzed by histology, and liver organoids were cultured and analyzed. We performed transcriptome analysis of Jag1Ndr/Ndr livers and livers from patients with Alagille syndrome, cross-referenced to the Human Protein Atlas, to identify commonly dysregulated pathways and biliary markers. We used species-specific transcriptome separation and ligand-receptor interaction assays to measure Notch signaling and the ability of JAG1Ndr to bind or activate Notch receptors. We studied signaling of JAG1 and JAG1Ndr via NOTCH 1, NOTCH2, and NOTCH3 and resulting gene expression patterns in parental and NOTCH1-expressing C2C12 cell lines. RESULTS: Jag1Ndr/Ndr mice had many features of Alagille syndrome, including eye, heart, and liver defects. Bile duct differentiation, morphogenesis, and function were dysregulated in newborn Jag1Ndr/Ndr mice, with aberrations in cholangiocyte polarity, but these defects improved in adult mice. Jag1Ndr/Ndr liver organoids collapsed in culture, indicating structural instability. Whole-transcriptome sequence analyses of liver tissues from mice and patients with Alagille syndrome identified dysregulated genes encoding proteins enriched at the apical side of cholangiocytes, including CFTR and SLC5A1, as well as reduced expression of IGF1. Exposure of Notch-expressing cells to JAG

Human liver cancer research currently lacks in vitro models that can faithfully recapitulate the pathophysiology of the original tumor. We recently described a novel, near-physiological organoid culture system, wherein primary human healthy liver cells form long-term expanding organoids that retain liver tissue function and genetic stability. Here we extend this culture system to the propagation of primary liver cancer (PLC) organoids from three of the most common PLC subtypes: hepatocellular carcinoma (HCC), cholangiocarcinoma (CC) and combined HCC/CC (CHC) tumors. PLC-derived organoid cultures preserve the histological architecture, gene expression and genomic landscape of the original tumor, allowing for discrimination between different tumor tissues and subtypes, even after long-term expansion in culture in the same medium conditions. Xenograft studies demonstrate that the tumorogenic potential, histological features and metastatic properties of PLC-derived organoids are preserved in vivo. PLC-derived organoids are amenable for biomarker identification and drug-screening testing and led to the identification of the ERK inhibitor SCH772984 as a potential therapeutic agent for primary liver cancer. We thus demonstrate the wide-ranging biomedical utilities of PLC-derived organoid models in furthering the understanding of liver cancer biology and in developing personalized-medicine approaches for the disease.

Hepatic steatosis is a highly prevalent liver disease, yet research is hampered by the lack of tractable cellular and animal models. Steatosis also occurs in cats, where it can cause severe hepatic failure. Previous studies demonstrate the potential of liver organoids for modeling genetic diseases. To examine the possibility of using organoids to model steatosis, we established a long-term feline liver organoid culture with adult liver stem cell characteristics and differentiation potential toward hepatocyte-like cells. Next, organoids from mouse, human, dog

Contains fulltext : 167652.pdf (publisher's version ) (Closed access), Autosomal dominant polycystic liver disease (ADPLD) is caused by variants in PRKCSH, SEC63, and LRP5, whereas autosomal dominant polycystic kidney disease is caused by variants in PKD1 and PKD2. Liver cyst development in these disorders is explained by somatic loss-of-heterozygosity (LOH) of the wild-type allele in the developing cyst. We hypothesize that we can use this mechanism to identify novel disease genes that reside in LOH regions. In this study, we aim to map abnormal genomic regions using high-density SNP microarrays to find novel PLD genes. We collected 46 cysts from 23 patients with polycystic or sporadic hepatic cysts, and analyzed DNA from those cysts using high-resolution microarray (n=24) or Sanger sequencing (n=22). We here focused on regions of homozygosity on the autosomes (>3.0 Mb) and large CNVs (>1.0 Mb). We found frequent LOH in PRKCSH (22/29) and PKD1/PKD2 (2/3) cysts of patients with known heterozygous germline variants in the respective genes. In the total cohort, 12/23 patients harbored abnormalities outside of familiar areas. In individual ADPLD cases, we identified germline events: a 2q13 complex rearrangement resulting in BUB1 haploinsufficiency, a 47XXX karyotype, chromosome 9q copy-number loss, and LOH on chromosome 3p. The latter region was overlapping with an LOH region identified in two other cysts. Unique germline and somatic abnormalities occur frequently in and outside of known genes underlying cysts. Each liver cyst has a unique genetic makeup. LOH driver gene BUB1 may imply germline causes of genetic instability in PLD.

Contains fulltext : 167652.pdf (publisher's version ) (Closed access), Autosomal dominant polycystic liver disease (ADPLD) is caused by variants in PRKCSH, SEC63, and LRP5, whereas autosomal dominant polycystic kidney disease is caused by variants in PKD1 and PKD2. Liver cyst development in these disorders is explained by somatic loss-of-heterozygosity (LOH) of the wild-type allele in the developing cyst. We hypothesize that we can use this mechanism to identify novel disease genes that reside in LOH regions. In this study, we aim to map abnormal genomic regions using high-density SNP microarrays to find novel PLD genes. We collected 46 cysts from 23 patients with polycystic or sporadic hepatic cysts, and analyzed DNA from those cysts using high-resolution microarray (n=24) or Sanger sequencing (n=22). We here focused on regions of homozygosity on the autosomes (>3.0 Mb) and large CNVs (>1.0 Mb). We found frequent LOH in PRKCSH (22/29) and PKD1/PKD2 (2/3) cysts of patients with known heterozygous germline variants in the respective genes. In the total cohort, 12/23 patients harbored abnormalities outside of familiar areas. In individual ADPLD cases, we identified germline events: a 2q13 complex rearrangement resulting in BUB1 haploinsufficiency, a 47XXX karyotype, chromosome 9q copy-number loss, and LOH on chromosome 3p. The latter region was overlapping with an LOH region identified in two other cysts. Unique germline and somatic abnormalities occur frequently in and outside of known genes underlying cysts. Each liver cyst has a unique genetic makeup. LOH driver gene BUB1 may imply germline causes of genetic instability in PLD.

Contains fulltext : 167652.pdf (publisher's version ) (Closed access), Autosomal dominant polycystic liver disease (ADPLD) is caused by variants in PRKCSH, SEC63, and LRP5, whereas autosomal dominant polycystic kidney disease is caused by variants in PKD1 and PKD2. Liver cyst development in these disorders is explained by somatic loss-of-heterozygosity (LOH) of the wild-type allele in the developing cyst. We hypothesize that we can use this mechanism to identify novel disease genes that reside in LOH regions. In this study, we aim to map abnormal genomic regions using high-density SNP microarrays to find novel PLD genes. We collected 46 cysts from 23 patients with polycystic or sporadic hepatic cysts, and analyzed DNA from those cysts using high-resolution microarray (n=24) or Sanger sequencing (n=22). We here focused on regions of homozygosity on the autosomes (>3.0 Mb) and large CNVs (>1.0 Mb). We found frequent LOH in PRKCSH (22/29) and PKD1/PKD2 (2/3) cysts of patients with known heterozygous germline variants in the respective genes. In the total cohort, 12/23 patients harbored abnormalities outside of familiar areas. In individual ADPLD cases, we identified germline events: a 2q13 complex rearrangement resulting in BUB1 haploinsufficiency, a 47XXX karyotype, chromosome 9q copy-number loss, and LOH on chromosome 3p. The latter region was overlapping with an LOH region identified in two other cysts. Unique germline and somatic abnormalities occur frequently in and outside of known genes underlying cysts. Each liver cyst has a unique genetic makeup. LOH driver gene BUB1 may imply germline causes of genetic instability in PLD.

Despite the enormous replication potential of the human liver, there are currently no culture systems available that sustain hepatocyte replication and/or function in vitro. We have shown previously that single mouse Lgr5+ liver stem cells can be expanded as epithelial organoids in vitro and can be differentiated into functional hepatocytes in vitro and in vivo. We now describe conditions allowing long-term expansion of adult bile duct-derived bipotent progenitor cells from human liver. The expanded cells are highly stable at the chromosome and structural level, while single base changes occur at very low rates. The cells can readily be converted into functional hepatocytes in vitro and upon transplantation in vivo. Organoids from alpha 1-antitrypsin deficiency and Alagille syndrome patients mirror the in vivo pathology. Clonal long-term expansion of primary adult liver stem cells opens up experimental avenues for disease modeling, toxicology studies, regenerative medicine, and gene therapy.

Leucine-rich repeat-containing G protein-coupled receptors 4-6 (LGR4-LGR6) are receptors for R-spondins, potent Wnt agonists that exert profound trophic effects on Wnt-driven stem cells compartments. We present crystal structures of a signaling-competent fragment of R-spondin 1 (Rspo1) at a resolution of 2.0 A and its complex with the LGR5 ectodomain at a resolution of 3.2 A. Ecto-LGR5 binds Rspo1 at its concave leucine-rich-repeat (LRR) surface, forming a dimeric 2:2 complex. Fully conserved residues on LGR4-LGR6 explain promiscuous binding of R-spondins. A phenylalanine clamp formed by Rspo1 Phe106 and Phe110 pinches Ala190 of LGR5 and is critical for binding. Mutations related to congenital anonychia reduce signaling, but not binding of Rspo1 to LGR5. Furthermore, antibody binding to the extended loop of the C-terminal LRR cap of LGR5 activates signaling in a ligand-independent manner. Thus, our data reveal binding of R-spondins to conserved sites on LGR4-LGR6 and, in analogy to FSHR and related receptors, suggest a direct signaling role for LGR4-LGR6 in addition to its formation of Wnt receptor and coreceptor complexes., Leucine-rich repeat-containing G protein-coupled receptors 4-6 (LGR4-LGR6) are receptors for R-spondins, potent Wnt agonists that exert profound trophic effects on Wnt-driven stem cells compartments. We present crystal structures of a signaling-competent fragment of R-spondin 1 (Rspo1) at a resolution of 2.0 A and its complex with the LGR5 ectodomain at a resolution of 3.2 A. Ecto-LGR5 binds Rspo1 at its concave leucine-rich-repeat (LRR) surface, forming a dimeric 2:2 complex. Fully conserved residues on LGR4-LGR6 explain promiscuous binding of R-spondins. A phenylalanine clamp formed by Rspo1 Phe106 and Phe110 pinches Ala190 of LGR5 and is critical for binding. Mutations related to congenital anonychia reduce signaling, but not binding of Rspo1 to LGR5. Furthermore, antibody binding to the extended loop of the C-terminal LRR cap of LGR5 activates signaling in a ligand-independent manner. Thus, our data reveal binding of R-spondins to conserved sites on LGR4-LGR6 and, in analogy to FSHR and related receptors, suggest a direct signaling role for LGR4-LGR6 in addition to its formation of Wnt receptor and coreceptor complexes.

Proliferation of the self-renewing epithelium of the gastric corpus occurs almost exclusively in the isthmus of the glands, from where cells migrate bidirectionally toward pit and base. The isthmus is therefore generally viewed as the stem cell zone. We find that the stem cell marker Troy is expressed at the gland base by a small subpopulation of fully differentiated chief cells. By lineage tracing with a Troy-eGFP-ires-CreERT2 allele, single marked chief cells are shown to generate entirely labeled gastric units over periods of months. This phenomenon accelerates upon tissue damage. Troy(+) chief cells can be cultured to generate long-lived gastric organoids. Troy marks a specific subset of chief cells that display plasticity in that they are capable of replenishing entire gastric units, essentially serving as quiescent "reserve" stem cells. These observations challenge the notion that stem cell hierarchies represent a "one-way street.", Proliferation of the self-renewing epithelium of the gastric corpus occurs almost exclusively in the isthmus of the glands, from where cells migrate bidirectionally toward pit and base. The isthmus is therefore generally viewed as the stem cell zone. We find that the stem cell marker Troy is expressed at the gland base by a small subpopulation of fully differentiated chief cells. By lineage tracing with a Troy-eGFP-ires-CreERT2 allele, single marked chief cells are shown to generate entirely labeled gastric units over periods of months. This phenomenon accelerates upon tissue damage. Troy(+) chief cells can be cultured to generate long-lived gastric organoids. Troy marks a specific subset of chief cells that display plasticity in that they are capable of replenishing entire gastric units, essentially serving as quiescent "reserve" stem cells. These observations challenge the notion that stem cell hierarchies represent a "one-way street."

Lgr5 marks adult stem cells in multiple adult organs and is a receptor for the Wnt-agonistic R-spondins (RSPOs). Intestinal, stomach and liver Lgr5(+) stem cells grow in 3D cultures to form ever-expanding organoids, which resemble the tissues of origin. Wnt signalling is inactive and Lgr5 is not expressed under physiological conditions in the adult pancreas. However, we now report that the Wnt pathway is robustly activated upon injury by partial duct ligation (PDL), concomitant with the appearance of Lgr5 expression in regenerating pancreatic ducts. In vitro, duct fragments from mouse pancreas initiate Lgr5 expression in RSPO1-based cultures, and develop into budding cyst-like structures (organoids) that expand five-fold weekly for >40 weeks. Single isolated duct cells can also be cultured into pancreatic organoids, containing Lgr5 stem/progenitor cells that can be clonally expanded. Clonal pancreas organoids can be induced to differentiate into duct as well as endocrine cells upon transplantation, thus proving their bi-potentiality., Lgr5 marks adult stem cells in multiple adult organs and is a receptor for the Wnt-agonistic R-spondins (RSPOs). Intestinal, stomach and liver Lgr5(+) stem cells grow in 3D cultures to form ever-expanding organoids, which resemble the tissues of origin. Wnt signalling is inactive and Lgr5 is not expressed under physiological conditions in the adult pancreas. However, we now report that the Wnt pathway is robustly activated upon injury by partial duct ligation (PDL), concomitant with the appearance of Lgr5 expression in regenerating pancreatic ducts. In vitro, duct fragments from mouse pancreas initiate Lgr5 expression in RSPO1-based cultures, and develop into budding cyst-like structures (organoids) that expand five-fold weekly for >40 weeks. Single isolated duct cells can also be cultured into pancreatic organoids, containing Lgr5 stem/progenitor cells that can be clonally expanded. Clonal pancreas organoids can be induced to differentiate into duct as well as endocrine cells upon transplantation, thus proving their bi-potentiality.

The Wnt target gene Lgr5 (leucine-rich-repeat-containing G-protein-coupled receptor 5) marks actively dividing stem cells in Wnt-driven, self-renewing tissues such as small intestine and colon, stomach and hair follicles. A three-dimensional culture system allows long-term clonal expansion of single Lgr5(+) stem cells into transplantable organoids (budding cysts) that retain many characteristics of the original epithelial architecture. A crucial component of the culture medium is the Wnt agonist RSPO1, the recently discovered ligand of LGR5. Here we show that Lgr5-lacZ is not expressed in healthy adult liver, however, small Lgr5-LacZ(+) cells appear near bile ducts upon damage, coinciding with robust activation of Wnt signalling. As shown by mouse lineage tracing using a new Lgr5-IRES-creERT2 knock-in allele, damage-induced Lgr5(+) cells generate hepatocytes and bile ducts in vivo. Single Lgr5(+) cells from damaged mouse liver can be clonally expanded as organoids in Rspo1-based culture medium over several months. Such clonal organoids can be induced to differentiate in vitro and to generate functional hepatocytes upon transplantation into Fah(-/-) mice. These findings indicate that previous observations concerning Lgr5(+) stem cells in actively self-renewing tissues can also be extended to damage-induced stem cells in a tissue with a low rate of spontaneous proliferation., The Wnt target gene Lgr5 (leucine-rich-repeat-containing G-protein-coupled receptor 5) marks actively dividing stem cells in Wnt-driven, self-renewing tissues such as small intestine and colon, stomach and hair follicles. A three-dimensional culture system allows long-term clonal expansion of single Lgr5(+) stem cells into transplantable organoids (budding cysts) that retain many characteristics of the original epithelial architecture. A crucial component of the culture medium is the Wnt agonist RSPO1, the recently discovered ligand of LGR5. Here we show that Lgr5-lacZ is not expressed in healthy adult liver, however, small Lgr5-LacZ(+) cells appear near bile ducts upon damage, coinciding with robust activation of Wnt signalling. As shown by mouse lineage tracing using a new Lgr5-IRES-creERT2 knock-in allele, damage-induced Lgr5(+) cells generate hepatocytes and bile ducts in vivo. Single Lgr5(+) cells from damaged mouse liver can be clonally expanded as organoids in Rspo1-based culture medium over several months. Such clonal organoids can be induced to differentiate in vitro and to generate functional hepatocytes upon transplantation into Fah(-/-) mice. These findings indicate that previous observations concerning Lgr5(+) stem cells in actively self-renewing tissues can also be extended to damage-induced stem cells in a tissue with a low rate of spontaneous proliferation.

Leucine-rich repeat-containing G protein-coupled receptors 4-6 (LGR4-LGR6) are receptors for R-spondins, potent Wnt agonists that exert profound trophic effects on Wnt-driven stem cells compartments. We present crystal structures of a signaling-competent fragment of R-spondin 1 (Rspo1) at a resolution of 2.0 A and its complex with the LGR5 ectodomain at a resolution of 3.2 A. Ecto-LGR5 binds Rspo1 at its concave leucine-rich-repeat (LRR) surface, forming a dimeric 2:2 complex. Fully conserved residues on LGR4-LGR6 explain promiscuous binding of R-spondins. A phenylalanine clamp formed by Rspo1 Phe106 and Phe110 pinches Ala190 of LGR5 and is critical for binding. Mutations related to congenital anonychia reduce signaling, but not binding of Rspo1 to LGR5. Furthermore, antibody binding to the extended loop of the C-terminal LRR cap of LGR5 activates signaling in a ligand-independent manner. Thus, our data reveal binding of R-spondins to conserved sites on LGR4-LGR6 and, in analogy to FSHR and related receptors, suggest a direct signaling role for LGR4-LGR6 in addition to its formation of Wnt receptor and coreceptor complexes., Leucine-rich repeat-containing G protein-coupled receptors 4-6 (LGR4-LGR6) are receptors for R-spondins, potent Wnt agonists that exert profound trophic effects on Wnt-driven stem cells compartments. We present crystal structures of a signaling-competent fragment of R-spondin 1 (Rspo1) at a resolution of 2.0 A and its complex with the LGR5 ectodomain at a resolution of 3.2 A. Ecto-LGR5 binds Rspo1 at its concave leucine-rich-repeat (LRR) surface, forming a dimeric 2:2 complex. Fully conserved residues on LGR4-LGR6 explain promiscuous binding of R-spondins. A phenylalanine clamp formed by Rspo1 Phe106 and Phe110 pinches Ala190 of LGR5 and is critical for binding. Mutations related to congenital anonychia reduce signaling, but not binding of Rspo1 to LGR5. Furthermore, antibody binding to the extended loop of the C-terminal LRR cap of LGR5 activates signaling in a ligand-independent manner. Thus, our data reveal binding of R-spondins to conserved sites on LGR4-LGR6 and, in analogy to FSHR and related receptors, suggest a direct signaling role for LGR4-LGR6 in addition to its formation of Wnt receptor and coreceptor complexes.

Proliferation of the self-renewing epithelium of the gastric corpus occurs almost exclusively in the isthmus of the glands, from where cells migrate bidirectionally toward pit and base. The isthmus is therefore generally viewed as the stem cell zone. We find that the stem cell marker Troy is expressed at the gland base by a small subpopulation of fully differentiated chief cells. By lineage tracing with a Troy-eGFP-ires-CreERT2 allele, single marked chief cells are shown to generate entirely labeled gastric units over periods of months. This phenomenon accelerates upon tissue damage. Troy(+) chief cells can be cultured to generate long-lived gastric organoids. Troy marks a specific subset of chief cells that display plasticity in that they are capable of replenishing entire gastric units, essentially serving as quiescent "reserve" stem cells. These observations challenge the notion that stem cell hierarchies represent a "one-way street.", Proliferation of the self-renewing epithelium of the gastric corpus occurs almost exclusively in the isthmus of the glands, from where cells migrate bidirectionally toward pit and base. The isthmus is therefore generally viewed as the stem cell zone. We find that the stem cell marker Troy is expressed at the gland base by a small subpopulation of fully differentiated chief cells. By lineage tracing with a Troy-eGFP-ires-CreERT2 allele, single marked chief cells are shown to generate entirely labeled gastric units over periods of months. This phenomenon accelerates upon tissue damage. Troy(+) chief cells can be cultured to generate long-lived gastric organoids. Troy marks a specific subset of chief cells that display plasticity in that they are capable of replenishing entire gastric units, essentially serving as quiescent "reserve" stem cells. These observations challenge the notion that stem cell hierarchies represent a "one-way street."

Lgr5 marks adult stem cells in multiple adult organs and is a receptor for the Wnt-agonistic R-spondins (RSPOs). Intestinal, stomach and liver Lgr5(+) stem cells grow in 3D cultures to form ever-expanding organoids, which resemble the tissues of origin. Wnt signalling is inactive and Lgr5 is not expressed under physiological conditions in the adult pancreas. However, we now report that the Wnt pathway is robustly activated upon injury by partial duct ligation (PDL), concomitant with the appearance of Lgr5 expression in regenerating pancreatic ducts. In vitro, duct fragments from mouse pancreas initiate Lgr5 expression in RSPO1-based cultures, and develop into budding cyst-like structures (organoids) that expand five-fold weekly for >40 weeks. Single isolated duct cells can also be cultured into pancreatic organoids, containing Lgr5 stem/progenitor cells that can be clonally expanded. Clonal pancreas organoids can be induced to differentiate into duct as well as endocrine cells upon transplantation, thus proving their bi-potentiality., Lgr5 marks adult stem cells in multiple adult organs and is a receptor for the Wnt-agonistic R-spondins (RSPOs). Intestinal, stomach and liver Lgr5(+) stem cells grow in 3D cultures to form ever-expanding organoids, which resemble the tissues of origin. Wnt signalling is inactive and Lgr5 is not expressed under physiological conditions in the adult pancreas. However, we now report that the Wnt pathway is robustly activated upon injury by partial duct ligation (PDL), concomitant with the appearance of Lgr5 expression in regenerating pancreatic ducts. In vitro, duct fragments from mouse pancreas initiate Lgr5 expression in RSPO1-based cultures, and develop into budding cyst-like structures (organoids) that expand five-fold weekly for >40 weeks. Single isolated duct cells can also be cultured into pancreatic organoids, containing Lgr5 stem/progenitor cells that can be clonally expanded. Clonal pancreas organoids can be induced to differentiate into duct as well as endocrine cells upon transplantation, thus proving their bi-potentiality.

The Wnt target gene Lgr5 (leucine-rich-repeat-containing G-protein-coupled receptor 5) marks actively dividing stem cells in Wnt-driven, self-renewing tissues such as small intestine and colon, stomach and hair follicles. A three-dimensional culture system allows long-term clonal expansion of single Lgr5(+) stem cells into transplantable organoids (budding cysts) that retain many characteristics of the original epithelial architecture. A crucial component of the culture medium is the Wnt agonist RSPO1, the recently discovered ligand of LGR5. Here we show that Lgr5-lacZ is not expressed in healthy adult liver, however, small Lgr5-LacZ(+) cells appear near bile ducts upon damage, coinciding with robust activation of Wnt signalling. As shown by mouse lineage tracing using a new Lgr5-IRES-creERT2 knock-in allele, damage-induced Lgr5(+) cells generate hepatocytes and bile ducts in vivo. Single Lgr5(+) cells from damaged mouse liver can be clonally expanded as organoids in Rspo1-based culture medium over several months. Such clonal organoids can be induced to differentiate in vitro and to generate functional hepatocytes upon transplantation into Fah(-/-) mice. These findings indicate that previous observations concerning Lgr5(+) stem cells in actively self-renewing tissues can also be extended to damage-induced stem cells in a tissue with a low rate of spontaneous proliferation., The Wnt target gene Lgr5 (leucine-rich-repeat-containing G-protein-coupled receptor 5) marks actively dividing stem cells in Wnt-driven, self-renewing tissues such as small intestine and colon, stomach and hair follicles. A three-dimensional culture system allows long-term clonal expansion of single Lgr5(+) stem cells into transplantable organoids (budding cysts) that retain many characteristics of the original epithelial architecture. A crucial component of the culture medium is the Wnt agonist RSPO1, the recently discovered ligand of LGR5. Here we show that Lgr5-lacZ is not expressed in healthy adult liver, however, small Lgr5-LacZ(+) cells appear near bile ducts upon damage, coinciding with robust activation of Wnt signalling. As shown by mouse lineage tracing using a new Lgr5-IRES-creERT2 knock-in allele, damage-induced Lgr5(+) cells generate hepatocytes and bile ducts in vivo. Single Lgr5(+) cells from damaged mouse liver can be clonally expanded as organoids in Rspo1-based culture medium over several months. Such clonal organoids can be induced to differentiate in vitro and to generate functional hepatocytes upon transplantation into Fah(-/-) mice. These findings indicate that previous observations concerning Lgr5(+) stem cells in actively self-renewing tissues can also be extended to damage-induced stem cells in a tissue with a low rate of spontaneous proliferation.

Most studies on TCF7L2 SNP variants in the pathogenesis of type 2 diabetes (T2D) focus on a role of the encoded transcription factor TCF4 in beta cells. Here, a mouse genetics approach shows that removal of TCF4 from beta cells does not affect their function, whereas manipulating TCF4 levels in the liver has major effects on metabolism. In Tcf7l2(-/-) mice, the immediate postnatal surge in liver metabolism does not occur. Consequently, pups die due to hypoglycemia. By combining chromatin immunoprecipitation with gene expression profiling, we identify a TCF4-controlled metabolic gene program that is acutely activated in the postnatal liver. In concordance, adult liver-specific Tcf7l2 knockout mice show reduced hepatic glucose production during fasting and display improved glucose homeostasis when maintained on high-fat diet. Furthermore, liver-specific TCF4 overexpression increases hepatic glucose production. These observations imply that TCF4 directly activates metabolic genes and that inhibition of Wnt signaling may be beneficial in metabolic disease., Most studies on TCF7L2 SNP variants in the pathogenesis of type 2 diabetes (T2D) focus on a role of the encoded transcription factor TCF4 in beta cells. Here, a mouse genetics approach shows that removal of TCF4 from beta cells does not affect their function, whereas manipulating TCF4 levels in the liver has major effects on metabolism. In Tcf7l2(-/-) mice, the immediate postnatal surge in liver metabolism does not occur. Consequently, pups die due to hypoglycemia. By combining chromatin immunoprecipitation with gene expression profiling, we identify a TCF4-controlled metabolic gene program that is acutely activated in the postnatal liver. In concordance, adult liver-specific Tcf7l2 knockout mice show reduced hepatic glucose production during fasting and display improved glucose homeostasis when maintained on high-fat diet. Furthermore, liver-specific TCF4 overexpression increases hepatic glucose production. These observations imply that TCF4 directly activates metabolic genes and that inhibition of Wnt signaling may be beneficial in metabolic disease.

Multipotent stem cells and their lineage-restricted progeny drive nephron formation within the developing kidney. Here, we document expression of the adult stem cell marker Lgr5 in the developing kidney and assess the stem/progenitor identity of Lgr5(+ve) cells via in vivo lineage tracing. The appearance and localization of Lgr5(+ve) cells coincided with that of the S-shaped body around embryonic day 14. Lgr5 expression remained restricted to cell clusters within developing nephrons in the cortex until postnatal day 7, when expression was permanently silenced. In vivo lineage tracing identified Lgr5 as a marker of a stem/progenitor population within nascent nephrons dedicated to generating the thick ascending limb of Henle's loop and distal convoluted tubule. The Lgr5 surface marker and experimental models described here will be invaluable for deciphering the contribution of early nephron stem cells to developmental defects and for isolating human nephron progenitors as a prerequisite to evaluating their therapeutic potential., Multipotent stem cells and their lineage-restricted progeny drive nephron formation within the developing kidney. Here, we document expression of the adult stem cell marker Lgr5 in the developing kidney and assess the stem/progenitor identity of Lgr5(+ve) cells via in vivo lineage tracing. The appearance and localization of Lgr5(+ve) cells coincided with that of the S-shaped body around embryonic day 14. Lgr5 expression remained restricted to cell clusters within developing nephrons in the cortex until postnatal day 7, when expression was permanently silenced. In vivo lineage tracing identified Lgr5 as a marker of a stem/progenitor population within nascent nephrons dedicated to generating the thick ascending limb of Henle's loop and distal convoluted tubule. The Lgr5 surface marker and experimental models described here will be invaluable for deciphering the contribution of early nephron stem cells to developmental defects and for isolating human nephron progenitors as a prerequisite to evaluating their therapeutic potential.

Most studies on TCF7L2 SNP variants in the pathogenesis of type 2 diabetes (T2D) focus on a role of the encoded transcription factor TCF4 in beta cells. Here, a mouse genetics approach shows that removal of TCF4 from beta cells does not affect their function, whereas manipulating TCF4 levels in the liver has major effects on metabolism. In Tcf7l2(-/-) mice, the immediate postnatal surge in liver metabolism does not occur. Consequently, pups die due to hypoglycemia. By combining chromatin immunoprecipitation with gene expression profiling, we identify a TCF4-controlled metabolic gene program that is acutely activated in the postnatal liver. In concordance, adult liver-specific Tcf7l2 knockout mice show reduced hepatic glucose production during fasting and display improved glucose homeostasis when maintained on high-fat diet. Furthermore, liver-specific TCF4 overexpression increases hepatic glucose production. These observations imply that TCF4 directly activates metabolic genes and that inhibition of Wnt signaling may be beneficial in metabolic disease., Most studies on TCF7L2 SNP variants in the pathogenesis of type 2 diabetes (T2D) focus on a role of the encoded transcription factor TCF4 in beta cells. Here, a mouse genetics approach shows that removal of TCF4 from beta cells does not affect their function, whereas manipulating TCF4 levels in the liver has major effects on metabolism. In Tcf7l2(-/-) mice, the immediate postnatal surge in liver metabolism does not occur. Consequently, pups die due to hypoglycemia. By combining chromatin immunoprecipitation with gene expression profiling, we identify a TCF4-controlled metabolic gene program that is acutely activated in the postnatal liver. In concordance, adult liver-specific Tcf7l2 knockout mice show reduced hepatic glucose production during fasting and display improved glucose homeostasis when maintained on high-fat diet. Furthermore, liver-specific TCF4 overexpression increases hepatic glucose production. These observations imply that TCF4 directly activates metabolic genes and that inhibition of Wnt signaling may be beneficial in metabolic disease.

Multipotent stem cells and their lineage-restricted progeny drive nephron formation within the developing kidney. Here, we document expression of the adult stem cell marker Lgr5 in the developing kidney and assess the stem/progenitor identity of Lgr5(+ve) cells via in vivo lineage tracing. The appearance and localization of Lgr5(+ve) cells coincided with that of the S-shaped body around embryonic day 14. Lgr5 expression remained restricted to cell clusters within developing nephrons in the cortex until postnatal day 7, when expression was permanently silenced. In vivo lineage tracing identified Lgr5 as a marker of a stem/progenitor population within nascent nephrons dedicated to generating the thick ascending limb of Henle's loop and distal convoluted tubule. The Lgr5 surface marker and experimental models described here will be invaluable for deciphering the contribution of early nephron stem cells to developmental defects and for isolating human nephron progenitors as a prerequisite to evaluating their therapeutic potential., Multipotent stem cells and their lineage-restricted progeny drive nephron formation within the developing kidney. Here, we document expression of the adult stem cell marker Lgr5 in the developing kidney and assess the stem/progenitor identity of Lgr5(+ve) cells via in vivo lineage tracing. The appearance and localization of Lgr5(+ve) cells coincided with that of the S-shaped body around embryonic day 14. Lgr5 expression remained restricted to cell clusters within developing nephrons in the cortex until postnatal day 7, when expression was permanently silenced. In vivo lineage tracing identified Lgr5 as a marker of a stem/progenitor population within nascent nephrons dedicated to generating the thick ascending limb of Henle's loop and distal convoluted tubule. The Lgr5 surface marker and experimental models described here will be invaluable for deciphering the contribution of early nephron stem cells to developmental defects and for isolating human nephron progenitors as a prerequisite to evaluating their therapeutic potential.

The study of gene function in endodermal epithelia such as of stomach, small intestine and colon relies heavily on transgenic approaches. Establishing such animal models is laborious, expensive and time-consuming. We present here a method based on Cre recombinase-inducible retrovirus vectors that allows the conditional manipulation of gene expression in primary mouse organoid culture systems., The study of gene function in endodermal epithelia such as of stomach, small intestine and colon relies heavily on transgenic approaches. Establishing such animal models is laborious, expensive and time-consuming. We present here a method based on Cre recombinase-inducible retrovirus vectors that allows the conditional manipulation of gene expression in primary mouse organoid culture systems.

The study of gene function in endodermal epithelia such as of stomach, small intestine and colon relies heavily on transgenic approaches. Establishing such animal models is laborious, expensive and time-consuming. We present here a method based on Cre recombinase-inducible retrovirus vectors that allows the conditional manipulation of gene expression in primary mouse organoid culture systems., The study of gene function in endodermal epithelia such as of stomach, small intestine and colon relies heavily on transgenic approaches. Establishing such animal models is laborious, expensive and time-consuming. We present here a method based on Cre recombinase-inducible retrovirus vectors that allows the conditional manipulation of gene expression in primary mouse organoid culture systems.

The study of gastric epithelial homeostasis and cancer has been hampered by the lack of stem cell markers and in vitro culture methods. The Wnt target gene Lgr5 marks stem cells in the small intestine, colon, and hair follicle. Here, we investigated Lgr5 expression in the stomach and assessed the stem cell potential of the Lgr5(+ve) cells by using in vivo lineage tracing. In neonatal stomach, Lgr5 was expressed at the base of prospective corpus and pyloric glands, whereas expression in the adult was predominantly restricted to the base of mature pyloric glands. Lineage tracing revealed these Lgr5(+ve) cells to be self-renewing, multipotent stem cells responsible for the long-term renewal of the gastric epithelium. With an in vitro culture system, single Lgr5(+ve) cells efficiently generated long-lived organoids resembling mature pyloric epithelium. The Lgr5 stem cell marker and culture method described here will be invaluable tools for accelerating research into gastric epithelial renewal, inflammation/infection, and cancer., The study of gastric epithelial homeostasis and cancer has been hampered by the lack of stem cell markers and in vitro culture methods. The Wnt target gene Lgr5 marks stem cells in the small intestine, colon, and hair follicle. Here, we investigated Lgr5 expression in the stomach and assessed the stem cell potential of the Lgr5(+ve) cells by using in vivo lineage tracing. In neonatal stomach, Lgr5 was expressed at the base of prospective corpus and pyloric glands, whereas expression in the adult was predominantly restricted to the base of mature pyloric glands. Lineage tracing revealed these Lgr5(+ve) cells to be self-renewing, multipotent stem cells responsible for the long-term renewal of the gastric epithelium. With an in vitro culture system, single Lgr5(+ve) cells efficiently generated long-lived organoids resembling mature pyloric epithelium. The Lgr5 stem cell marker and culture method described here will be invaluable tools for accelerating research into gastric epithelial renewal, inflammation/infection, and cancer.

Cancer in the 21st century has become the number one cause of death in developed countries. Although much progress has been made in improving patient survival, tumour relapse is one of the important causes of cancer treatment failure. An early observation in the study of cancer was the heterogeneity of tumours. Traditionally, this was explained by a combination of genomic instability of tumours and micro environmental factors leading to diverse phenotypical characteristics. It was assumed that cells in a tumour have an equal capacity to propagate the cancer. This model is currently known as the stochastic model. Recently, the Cancer stem cell model has been proposed to explain the heterogeneity of a tumour and its progression. According to this model, the heterogeneity of tumours is the result of aberrant differentiation of tumour cells into the cells of the tissue the tumour originated from. Tumours were suggested to contain stem cell-like cells, the cancer stem cells or tumour-initiating cells, which are uniquely capable of propagating a tumour much like normal stem cells fuel proliferation and differentiation in normal tissue. In this review we discuss the normal stem cell biology of the stomach and intestine followed by both the stochastic and cancer stem cell models in light of recent findings in the gastric and intestinal systems. The molecular pathways underlying normal and tumourigenic growth have been well studied, and recently the stem cells of the stomach and intestine have been identified. Furthermore, intestinal stem cells were identified as the cells-of-origin of colon cancer upon loss of the tumour suppressor APC. Lastly, several studies have proposed the positive identification of a cancer stem cell of human colon cancer. At the end we compare the cancer stem cell model and the stochastic model. We conclude that clonal evolution of tumour cells resulting from genetic mutations underlies tumour initiation and progression in both cancer models. This im, Cancer in the 21st century has become the number one cause of death in developed countries. Although much progress has been made in improving patient survival, tumour relapse is one of the important causes of cancer treatment failure. An early observation in the study of cancer was the heterogeneity of tumours. Traditionally, this was explained by a combination of genomic instability of tumours and micro environmental factors leading to diverse phenotypical characteristics. It was assumed that cells in a tumour have an equal capacity to propagate the cancer. This model is currently known as the stochastic model. Recently, the Cancer stem cell model has been proposed to explain the heterogeneity of a tumour and its progression. According to this model, the heterogeneity of tumours is the result of aberrant differentiation of tumour cells into the cells of the tissue the tumour originated from. Tumours were suggested to contain stem cell-like cells, the cancer stem cells or tumour-initiating cells, which are uniquely capable of propagating a tumour much like normal stem cells fuel proliferation and differentiation in normal tissue. In this review we discuss the normal stem cell biology of the stomach and intestine followed by both the stochastic and cancer stem cell models in light of recent findings in the gastric and intestinal systems. The molecular pathways underlying normal and tumourigenic growth have been well studied, and recently the stem cells of the stomach and intestine have been identified. Furthermore, intestinal stem cells were identified as the cells-of-origin of colon cancer upon loss of the tumour suppressor APC. Lastly, several studies have proposed the positive identification of a cancer stem cell of human colon cancer. At the end we compare the cancer stem cell model and the stochastic model. We conclude that clonal evolution of tumour cells resulting from genetic mutations underlies tumour initiation and progression in both cancer models. This im

The study of gastric epithelial homeostasis and cancer has been hampered by the lack of stem cell markers and in vitro culture methods. The Wnt target gene Lgr5 marks stem cells in the small intestine, colon, and hair follicle. Here, we investigated Lgr5 expression in the stomach and assessed the stem cell potential of the Lgr5(+ve) cells by using in vivo lineage tracing. In neonatal stomach, Lgr5 was expressed at the base of prospective corpus and pyloric glands, whereas expression in the adult was predominantly restricted to the base of mature pyloric glands. Lineage tracing revealed these Lgr5(+ve) cells to be self-renewing, multipotent stem cells responsible for the long-term renewal of the gastric epithelium. With an in vitro culture system, single Lgr5(+ve) cells efficiently generated long-lived organoids resembling mature pyloric epithelium. The Lgr5 stem cell marker and culture method described here will be invaluable tools for accelerating research into gastric epithelial renewal, inflammation/infection, and cancer., The study of gastric epithelial homeostasis and cancer has been hampered by the lack of stem cell markers and in vitro culture methods. The Wnt target gene Lgr5 marks stem cells in the small intestine, colon, and hair follicle. Here, we investigated Lgr5 expression in the stomach and assessed the stem cell potential of the Lgr5(+ve) cells by using in vivo lineage tracing. In neonatal stomach, Lgr5 was expressed at the base of prospective corpus and pyloric glands, whereas expression in the adult was predominantly restricted to the base of mature pyloric glands. Lineage tracing revealed these Lgr5(+ve) cells to be self-renewing, multipotent stem cells responsible for the long-term renewal of the gastric epithelium. With an in vitro culture system, single Lgr5(+ve) cells efficiently generated long-lived organoids resembling mature pyloric epithelium. The Lgr5 stem cell marker and culture method described here will be invaluable tools for accelerating research into gastric epithelial renewal, inflammation/infection, and cancer.

Cancer in the 21st century has become the number one cause of death in developed countries. Although much progress has been made in improving patient survival, tumour relapse is one of the important causes of cancer treatment failure. An early observation in the study of cancer was the heterogeneity of tumours. Traditionally, this was explained by a combination of genomic instability of tumours and micro environmental factors leading to diverse phenotypical characteristics. It was assumed that cells in a tumour have an equal capacity to propagate the cancer. This model is currently known as the stochastic model. Recently, the Cancer stem cell model has been proposed to explain the heterogeneity of a tumour and its progression. According to this model, the heterogeneity of tumours is the result of aberrant differentiation of tumour cells into the cells of the tissue the tumour originated from. Tumours were suggested to contain stem cell-like cells, the cancer stem cells or tumour-initiating cells, which are uniquely capable of propagating a tumour much like normal stem cells fuel proliferation and differentiation in normal tissue. In this review we discuss the normal stem cell biology of the stomach and intestine followed by both the stochastic and cancer stem cell models in light of recent findings in the gastric and intestinal systems. The molecular pathways underlying normal and tumourigenic growth have been well studied, and recently the stem cells of the stomach and intestine have been identified. Furthermore, intestinal stem cells were identified as the cells-of-origin of colon cancer upon loss of the tumour suppressor APC. Lastly, several studies have proposed the positive identification of a cancer stem cell of human colon cancer. At the end we compare the cancer stem cell model and the stochastic model. We conclude that clonal evolution of tumour cells resulting from genetic mutations underlies tumour initiation and progression in both cancer models. This im, Cancer in the 21st century has become the number one cause of death in developed countries. Although much progress has been made in improving patient survival, tumour relapse is one of the important causes of cancer treatment failure. An early observation in the study of cancer was the heterogeneity of tumours. Traditionally, this was explained by a combination of genomic instability of tumours and micro environmental factors leading to diverse phenotypical characteristics. It was assumed that cells in a tumour have an equal capacity to propagate the cancer. This model is currently known as the stochastic model. Recently, the Cancer stem cell model has been proposed to explain the heterogeneity of a tumour and its progression. According to this model, the heterogeneity of tumours is the result of aberrant differentiation of tumour cells into the cells of the tissue the tumour originated from. Tumours were suggested to contain stem cell-like cells, the cancer stem cells or tumour-initiating cells, which are uniquely capable of propagating a tumour much like normal stem cells fuel proliferation and differentiation in normal tissue. In this review we discuss the normal stem cell biology of the stomach and intestine followed by both the stochastic and cancer stem cell models in light of recent findings in the gastric and intestinal systems. The molecular pathways underlying normal and tumourigenic growth have been well studied, and recently the stem cells of the stomach and intestine have been identified. Furthermore, intestinal stem cells were identified as the cells-of-origin of colon cancer upon loss of the tumour suppressor APC. Lastly, several studies have proposed the positive identification of a cancer stem cell of human colon cancer. At the end we compare the cancer stem cell model and the stochastic model. We conclude that clonal evolution of tumour cells resulting from genetic mutations underlies tumour initiation and progression in both cancer models. This im